US7977457B2 - Fusion proteins, uses thereof and processes for producing same - Google Patents

Fusion proteins, uses thereof and processes for producing same Download PDF

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US7977457B2
US7977457B2 US11/804,541 US80454107A US7977457B2 US 7977457 B2 US7977457 B2 US 7977457B2 US 80454107 A US80454107 A US 80454107A US 7977457 B2 US7977457 B2 US 7977457B2
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cells
peptide
mesothelin
scfv
hla
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US20080014208A1 (en
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Yoram Reiter
Roy Noy
Kfir Oved
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Technion Research and Development Foundation Ltd
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Teva Pharmaceutical Industries Ltd
Technion Research and Development Foundation Ltd
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Priority to US14/526,667 priority patent/US20150152161A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • Tumor evasion from immune response is a well established phenomenon demonstrated in numerous studies and is caused by a wide variety of suggested mechanisms (1-4). Among these mechanisms are: the production of suppressive cytokines, the loss of immunodominant peptides, the resistance to killing mechanisms (apoptosis), and the loss of MHC class I (1-4).
  • One of the evasion mechanisms shown to be strongly correlated with tumor progression is the loss or down regulation of MHC class I molecules. This evasion mechanism is abundant in many tumors and can result from a number of different mutations.
  • MHC class I loading and presentation route including loss of beta-2-microglobulin, TAP1/TAP2 mutations, LMP mutations, loss of heterozygocity in the MHC genes, and down regulation of specific MHC alleles.
  • the second strategy adoptive cell transfer, has recently shown impressive results in metastatic melanoma patients in which highly selected, tumor-reactive T-cells against different over-expressed self-derived differentiation antigens were isolated, expended ex-vivo and reintroduced to the patients.
  • This approach a persistent clonal repopulation of T-cells, proliferation in vivo, functional activity, and trafficking to tumor sites were demonstrated (12-14).
  • a new immunotherapeutic approach recently presented takes advantage of two well-established areas: (i) the known effectiveness of CD8+ cytotoxic T-lymphocytes in the elimination of cells presenting highly immunogenic MHC/peptide complexes, and (ii) the tumor-specific cell surface antigens targeting via recombinant fragments of antibodies, mainly single chain Fv fragments (scFvs).
  • This approach utilizes a recombinant fusion protein composed of two functionally distinct entities: (i) a single-chain MHC class I molecule that carries a highly immunogenic tumor or viral-derived peptide, and (ii) a tumor-specific, high-affinity scFv fragment (15).
  • the MHC class I-restricted CD8+ cytotoxic T-cell (CTL) effector arm of the adaptive immune response is best equipped to recognize tumor cells as foreign and initiate the cascade of events resulting in tumor destruction.
  • CTL cytotoxic T-cell
  • a recombinant molecule was constructed in which a single-chain MHC is specifically targeted to tumor cells through its fusion to cancer specific-recombinant antibody fragments or a ligand that binds to receptors expressed by tumor cells.
  • This invention provides a fusion protein comprising consecutive amino acids which, beginning at the amino terminus of the protein, correspond to consecutive amino acids present in (i) a cytomegalovirus human MHC-restricted peptide, (ii) a first peptide linker, (iii) a human ⁇ -2 microglobulin, (iv) a second peptide linker, (v) a HLA-A2 chain of a human MHC class I molecule, (vi) a third peptide linker, (vii) a variable region from a heavy chain of a scFv fragment of an antibody, and (viii) a variable region from a light chain of such scFv fragment, wherein the consecutive amino acids which correspond to (vii) and (viii) are bound together directly by a peptide bond or by consecutive amino acids which correspond to a fourth peptide linker and the scFv fragment is derived from an antibody which specifically binds to mesothelin.
  • compositions comprising the fusion protein and a carrier.
  • This invention further provides a nucleic acid construct encoding a fusion protein comprising consecutive amino acids which, beginning at the amino terminus of the protein, correspond to consecutive amino acids present in (i) a cytomegalovirus human MHC-restricted peptide, (ii) a first peptide linker, (iii) a human ⁇ -2 microglobulin, (iv) a second peptide linker, (v) a HLA-A2 chain of a human MHC class I molecule, (vi) a third peptide linker, (vii) a variable region from a heavy chain of a scFv fragment of an antibody, and (viii) a variable region from a light chain of such scFv fragment, wherein the consecutive amino acids which correspond to (vii) and (viii) are bound together directly by a peptide bond or by consecutive amino acids which correspond to a fourth peptide linker and the scFv fragment is derived from an antibody which specifically binds to
  • This invention still further provides an isolated preparation of bacterially-expressed inclusion bodies comprising over 30 percent by weight of a fusion protein in accordance with the invention.
  • This invention also provides a process for producing a fusion protein comprising culturing a transformed cell comprising the fusion protein, so that the fusion protein is expressed, and recovering the fusion protein so expressed.
  • This invention further provides a method of selectively killing a tumor cell which comprises contacting the cell with the fusion protein of the invention in an amount effective to initiate a CTL-mediated immune response against the tumor cell so as to thereby kill the tumor cell.
  • this invention further provides a method of treating a tumor cell which expresses mesothelin on its surface, which comprises contacting the tumor cell with the fusion protein according to the invention in an amount effective to initiate a CTL-mediated immune response against the tumor cell so as to thereby treat the tumor cell.
  • a single-chain MHC molecule composed of ⁇ 2 microglobulin fused to the ⁇ 1, ⁇ 2 and ⁇ 3 domains of HLA-A2 via a short peptide linker (15 amino acids) was fused to the scFv SS1 which targets mesothelin.
  • a 9 amino acids peptide derived from the CMV pp65 protein NLVPMVATV was fused to the N-terminus of the scHLA-A2/SS1 (scFv) fusion protein via a 20 amino acid linker GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:6).
  • the fusion protein was expressed in E. coli and functional molecules were produced by in vitro refolding in the presence of CMV/scHLA-A2/SS1 (scFv).
  • Flow cytometry studies revealed the ability to decorate antigen-positive, HLA-A2-negative human tumor cells with HLA-A2-peptide complexes in a manner that was entirely dependent upon the specificity of the targeting antibody fragment.
  • CMV/scHLA-A2/SS1 (scFv)-mediated coating of target tumor cells made them susceptible for efficient and specific HLA-A2-restricted, CMV peptide-specific CTL-mediated lysis.
  • a novel strategy was developed to re-target class I MHC-peptide complexes on the surface of tumor cells in a way that is independent of the extent of class I MHC expression by the target tumor cells.
  • a molecule with two arms was employed.
  • One arm, the targeting moiety comprises tumor-specific recombinant fragments of antibodies directed to tumor or differentiation antigens which have been used for many years to target radioisotopes, toxins or drugs to cancer cells.
  • the second effector arm is a single-chain MHC molecule (scMHC) composed of human ⁇ 2-microglobulin linked to the three extracellular domains of the HLA-A2 heavy chain (24, 25, WO 01/72768).
  • the new molecule is expressed efficiently in E. coli and produced, for example, by in vitro refolding in the presence of HLA-A2-CMV peptides.
  • This approach renders the target tumor cells susceptible to lysis by cytotoxic T-cells regardless of their MHC expression level and thus may be employed as a new approach to potentiate CTL-mediated anti-tumor immunity.
  • This novel approach will lead to the development of a new class of recombinant therapeutic agents capable of selective killing and elimination of tumor cells utilizing natural cognate MHC ligands and CTL-based cytotoxic mechanisms.
  • FIGS. 1A-B are identical to FIGS. 1A-B.
  • FIG. 1A illustrates the C-Terminus of the scHLA-A2 fused to the N-terminus of SS1 (scFv) via a 4 amino acid linker.
  • FIG. 1B illustrates that the CMV pp65 peptide, i.e. NLVPMVATV (SEQ ID NO:4) was fused to the N-terminus of the scHLA-A2/SS1 (scFv) via a 20 amino acid linker GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:6).
  • FIG. 3A shows the SDS/PAGE analysis of isolated inclusion bodies.
  • FIG. 3B shows the SDS/PAGE analysis of Compound A after purification on ion-exchange chromatography.
  • FIGS. 5A-D are identical to FIGS. 5A-D.
  • FIG. 5A-B demonstrates the flow cytometry analysis of the binding of Compound A to mesothelin-positive HLA-A2-negative A431K5 cells and mesothelin-negative HLA-A2-negative A431 cells.
  • FIG. 5A shows the binding of the K1 mAb (31, 32) to A431K5 cells
  • FIG. 5B shows the absence of binding of the K1 mAb to A431 cells.
  • FIG. 5C shows the binding of Compound A to A431K5 cells
  • FIG. 5D shows the absence of binding of Compound A to A431 cells.
  • the binding was monitored using anti-HLA-A2 specific antibody BB7.2 (35) and a FITC-labeled secondary antibody.
  • FIG. 6A the mesothelin-transfected A431K5 cells and the parental mesothelin-negative A431 cells were incubated with Compound A (10 ⁇ g) and CMV specific CTLs in a [S 35 ] methionine release assay.
  • FIG. 6B demonstrates dose-dependent activity of Compound A when mesothelin-transfected A431K5 cells and the parental mesothelin-negative A431 cells were incubated with different concentrations of Compound A and CMV-specific CTLs in a [S 35 ] methionine release assay.
  • FIG. 9A shows SDS/PAGE analysis of isolated inclusion bodies.
  • FIG. 9B shows SDS/PAGE analysis of Compound B after purification on ion-exchange chromatography.
  • FIGS. 10A-F demonstrate the flow cytometry analysis of the binding of Compound B to mesothelin-positive HLA-A2-negative A431K5 cells and mesothelin-negative HLA-A2-negative A431 cells.
  • FIG. 10A shows the binding of K1 mAb to A431K5 cells
  • FIG. 10B shows the lack of binding of K1 mAb to A431 cells (B).
  • FIG. 10C shows the binding of Compound B to A431K5 cells
  • FIG. 10D shows the lack of binding of Compound B to A431 cells.
  • FIG. 10E shows the comparison between the binding of Compound A and Compound B to A431K5 cells
  • FIG. 10F shows the lack of binding of Compound A and Compound B to A431 cells.
  • the binding was monitored using anti-HLA-A2 specific antibody BB7.2 and a FITC-labeled secondary antibody.
  • FIG. 11A mesothelin-transfected A431K5 cells and the parental mesothelin-negative A431 cells were incubated with Compound B (10 ⁇ g) and CMV-specific CTLs in a [S 35 ]methionine release assay.
  • FIG. 11B demonstrates dose-dependent activity of Compound B, when mesothelin-transfected A431K5 cells and the parental mesothelin-negative A431 cells were incubated with different concentrations of Compound A and CMV-specific CTLs in a [S 35 ] methionine release assay
  • FIG. 13A shows the C-terminus of the scHLA-A2 fused to the N-terminus of scFv via 4 amino acid linker.
  • FIG. 13B shows the M158-66 peptide fused to the N-terminus of the scHLA-A2/SS1 (scFv) via a 15 amino acid linker GGGGSGGGGSGGGGS (SEQ ID NO:8).
  • Nucleic acid sequence encoding the M1cov/scHLA-A2/SS1 (scFv) fusion protein (SEQ ID NO:23).
  • FIG. 15A shows the SDS/PAGE analysis of isolated inclusion bodies.
  • FIG. 15B shows the SDS/PAGE analysis of M1-cov/scHLA-A2/SS1 (scFv) fusion protein after purification on ion-exchange chromatography.
  • M1-cov/scHLA-A2/SS1 (scFv) fusion protein to recombinant Mesothelin.
  • Mesothelin was immobilized onto immuno-plates and dose-dependent binding of M1-cov/scHLA-A2/SS1 (scFv) was monitored by conformation sensitive mAb (W6).
  • FIGS. 17A-D are identical to FIGS. 17A-D.
  • FIGS. 17A-D show flow cytometry analysis of the binding of M1-cov/scHLA-A2/SS1 (scFv) to mesothelin-positive HLA-A2-negative A431K5 cells and mesothelin-negative HLA-A2-negative A431 cells.
  • FIG. 17A shows the binding of K1 mAb to A431K5 cells
  • FIG. 17B shows the absence of binding of K1 mAb to A431 cells.
  • FIG. 17C shows the binding of M1-cov/scHLA-A2/SS1 (scFv) fusion protein to A431K5 cells
  • FIG. 17D shows the absence of binding of M1-cov/scHLA-A2/SS1 (scFv) fusion protein to A431 cells.
  • the binding was monitored using anti-HLA-A2 specific antibody BB7.2 and a FITC-labeled secondary antibody.
  • This invention provides a fusion protein comprising consecutive amino acids which, beginning at the amino terminus of the protein, correspond to consecutive amino acids present in (i) a cytomegalovirus human MHC-restricted peptide, (ii) a first peptide linker, (iii) a human ⁇ -2 microglobulin, (iv) a second peptide linker, (v) a HLA-A2 chain of a human MHC class I molecule, (vi) a third peptide linker, (vii) a variable region from a heavy chain of a scFv fragment of an antibody, and (viii) a variable region from a light chain of such scFv fragment, wherein the consecutive amino acids which correspond to (vii) and (viii) are bound together directly by a peptide bond or by consecutive amino acids which correspond to a fourth peptide linker and the scFv fragment is derived from an antibody which specifically binds to mesothelin.
  • the first peptide linker has the amino acid sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:6).
  • the second peptide linker has the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO:8).
  • the third peptide linker has the amino acid sequence ASGG (SEQ ID NO:10).
  • the fourth peptide linker has the amino acid sequence GVGGSGGGGSGGGGS (SEQ ID NO:19).
  • the cytomegalovirus human MHC-restricted peptide has the amino acid sequence NLVPMVATV (SEQ ID NO:4).
  • first peptide linker refers to peptides composed of a monomeric peptide whose amino acid sequence is GXGGS (SEQ ID NO:20) or a multimer thereof, wherein X may be any amino acid.
  • These peptide linkers may be a multimer of 2-10 of such monomeric peptide.
  • each monomeric peptide may be the same as or different from other monomeric peptide in the multimer depending on the identity of amino acid X.
  • X in the monomeric peptide is the amino acid valine (V).
  • X in the monomeric peptide is the amino acid glycine (G).
  • the peptide linker comprises a multimer of three or four monomeric peptides, particularly a multimer of three monomeric peptides in which the most N-terminal X is the amino acid V, and the second and third X are the amino acid G.
  • sequence of the consecutive amino acids corresponding to (vii), followed by the fourth peptide linker, followed by (viii) is set forth in SEQ ID NO:12.
  • the consecutive amino acids of the fusion protein, Compound A have the amino acid sequence set forth in SEQ ID NO:2.
  • This invention also provides a composition comprising a fusion protein in accordance with the invention and a carrier.
  • the fusion protein is present in the composition in a therapeutically effective amount and the carrier is a pharmaceutically acceptable carrier.
  • This invention also provides a nucleic acid construct encoding a fusion protein comprising consecutive amino acids which, beginning at the amino terminus of the protein, correspond to consecutive amino acids present in (i) a cytomegalovirus human MHC-restricted peptide, (ii) a first peptide linker, (iii) a human ⁇ -2 microglobulin, (iv) a second peptide linker, (v) a HLA-A2 chain of a human MHC class I molecule, (vi) a third peptide linker, (vii) a variable region from a heavy chain of a scFv fragment of an antibody, and (viii) a variable region from a light chain of such scFv fragment, wherein the consecutive amino acids which correspond to (vii) and (viii) are bound together directly by a peptide bond or by consecutive amino acids which correspond to a fourth peptide linker and the scFv fragment is derived from an antibody which specifically binds to
  • This invention also provides a vector comprising the nucleic acid construct of the invention.
  • vectors are plasmids, viruses, phages, and the like.
  • This invention further provides an expression vector comprising the nucleic acid construct of the invention and a promoter operatively linked thereto.
  • This invention also provides a transformed cell comprising a vector according to the invention.
  • the transformed cell may be a eukaryotic cell, e.g. one selected from the group consisting of a mammalian cell, an insect cell, a plant cell, a yeast cell and a protozoa cell.
  • the transformed cell may be a bacterial cell.
  • This invention provides an isolated preparation of bacterially-expressed inclusion bodies comprising over 30 percent by weight of a fusion protein according to the invention.
  • This invention also provides a process for producing a fusion protein comprising culturing the transformed cell of the invention so that the fusion protein is expressed, and recovering the fusion protein so expressed.
  • the recovery of the fusion protein comprises subjecting the expressed fusion protein to size exclusion chromatography.
  • the fusion protein is expressed in inclusion bodies.
  • the process further comprises treating the inclusion bodies so as to separate and refold the fusion protein and thereby produce the fusion protein in active form.
  • treating of the inclusion bodies to separate the fusion protein therefrom comprises contacting the inclusion bodies with a denaturing agent.
  • an “active form” of the fusion protein means a three dimensional conformation of the fusion protein which permits the fusion protein to specifically bind to mesothelin when mesothelin is present on the surface of a tumor cell.
  • This invention also provides a method of selectively killing a tumor cell, which comprises contacting the cell with the fusion protein of the invention in an amount effective to initiate a CTL-mediated immune response against the tumor cell so as to thereby kill the tumor cell.
  • the tumor cell is in a patient and the contacting is effected by administering the fusion protein to the patient.
  • This invention further provides a method of treating a tumor cell which expresses mesothelin on its surface, which comprises contacting the tumor cell with the fusion protein according to the invention in an amount effective to initiate a CTL-mediated immune response against the tumor cell so as to thereby treat the tumor cell.
  • the tumor cell is present in a solid tumor.
  • the solid tumor is a tumor associated with ovarian, lung, pancreatic or head/neck cancer, or mesothelioma.
  • the present invention provides (i) novel fusion proteins; (ii) processes of preparing same; (iii) nucleic acid constructs encoding same; and (iv) methods of using same for selective killing of cells, cancer cells in particular.
  • Tumor progression is often associated with the secretion of immune-suppressive factors and/or the down-regulation of MHC class I antigen-presentation functions (2). Even when a specific CTL response is demonstrated in patients, this response is low because the anti-tumor CTL population is rare, very infrequent, and in some cases the CLTs are not functional or anergic (26). Moreover, it is well-established that the number of MHC-peptide complexes on the surface of tumor cells that present a particular tumor-associated peptide is low (27).
  • the present invention provides a new approach to circumvent this problem. While reducing the present invention to practice, tumor-specific targeting of class I MHC-peptide complexes on tumor cells was shown to be an effective and efficient strategy to render HLA-A2-negative cells susceptible to lysis by relevant HLA-A2-restricted CTLs. This new strategy of redirecting CTLs against tumor cells takes advantage of the use of recombinant anti-mesothelin antibody fragment and CMV ligand that can localize on malignant cells that express a tumor with a relatively high degree of specificity.
  • the anti-mesothelin antibody targeting fragment and CMV ligand are fused to a single-chain HLA-A2 molecule that can be folded efficiently and functionally.
  • results presented herein provide a clear demonstration of the usefulness of the approach of the present invention to recruit active CTLs for tumor cell killing via cancer-specific antibody or ligand guided targeting of scMHC-peptide complexes. These results pave the way for the development of a new immunotherapeutic approach based on naturally occurring cellular immune responses which are redirected against the tumor cells.
  • the fusion protein of the present invention or portions thereof can be prepared by several ways, including solid phase protein synthesis.
  • at least major portions of the molecules e.g., the scHLA-A2 domain (with or without the CMV peptide) and the scFV domain are generated by translation of a respective nucleic acid construct or constructs encoding the molecule.
  • one to three open reading frames are required to synthesize the molecules of FIG. 1B via translation.
  • These open reading frames can reside on a single, two or three nucleic acid molecules.
  • a single nucleic acid construct can carry one, two or all three open reading frames.
  • One to three cis-acting regulatory sequences can be used to control the expression of the one to three open reading frames.
  • a single cis-acting regulatory sequence can control the expression of one, two or three open reading frames, in a cistrone-like manner.
  • three independent cis-acting regulatory sequences can be used to control the expression of the three open reading frames. Other combinations are also envisaged.
  • the open reading frames and the cis-acting regulatory sequences can be carried by one to three nucleic acid molecules.
  • each open reading frame and its cis-acting regulatory sequence are carried by a different nucleic acid molecule, or all of the open reading frames and their associated cis-acting regulatory sequences are carried by a single nucleic acid molecule.
  • Other combinations are also envisaged.
  • Fusion protein can be effected by transformation/transfection and/or co-transformation/co-transfection of a single cell or a plurality of cells with any of the nucleic acid molecules, serving as transformation/transfection vectors (e.g., as plasmids, phages, phagemids or viruses).
  • transformation/transfection vectors e.g., as plasmids, phages, phagemids or viruses.
  • the fusion protein whose amino acid sequence is set forth in SEQ ID NO:2 and includes the N-terminal amino acid methionine, likely represents the fusion protein as expressed in a bacterial cell.
  • the N-terminal methionine may be cleaved and removed.
  • fusion proteins in accordance with this invention encompass both those with, and those without, a N-terminal methionine.
  • amino acid sequence of expressed fusion proteins according to the invention may include or not include such N-terminal methionine depending on the type of cells in which the proteins are expressed.
  • the linker peptide is selected of an amino acid sequence which is inherently flexible, such that the polypeptides connected thereby independently and natively fold following expression thereof, thus facilitating the formation of a functional or active single chain (sc) human ⁇ 2 M/HLA complex, antibody targeting or human ⁇ 2 M/HLA-CMV restricted antigen complex.
  • sc single chain
  • any of the nucleic acid constructs described herein comprise at least one cis-acting regulatory sequence operably linked to the coding polynucleotides therein.
  • the cis-acting regulatory sequence is functional in bacteria.
  • the cis-acting regulatory sequence is functional in yeast.
  • the cis-acting regulatory sequence is functional in animal cells.
  • the cis acting regulatory sequence is functional in plant cells.
  • the cis-acting regulatory sequence can include a promoter sequence and additional transcriptional or a translational enhancer sequences all of which serve for facilitating the expression of the polynucleotides when introduced into a host cell.
  • promoters are described hereinbelow in context of various eukaryotic and prokaryotic expression systems and in the examples section which follows.
  • a single cis-acting regulatory sequence can be utilized in a nucleic acid construct to direct transcription of a single transcript which includes one or more open reading frames.
  • an internal ribosome entry site IVS
  • IVS internal ribosome entry site
  • the construct or constructs employed must be configured such that the levels of expression of the independent polypeptides are optimized, so as to obtain highest proportions of the final product.
  • a promoter being an example of a cis-acting regulatory sequence
  • a promoter utilized by the nucleic acid construct(s) of the present invention is a strong constitutive promoter such that high levels of expression are attained for the polynucleotides following host cell transformation.
  • high levels of expression can also be effected by transforming the host cell with a high copy number of the nucleic acid construct(s), or by utilizing cis acting sequences which stabilize the resultant transcript and as such decrease the degradation or “turn-over” of such a transcript.
  • transformed cell describes a cell into which an exogenous nucleic acid sequence is introduced to thereby stably or transiently genetically alter the host cell. It may occur under natural or artificial conditions using various methods well known in the art some of which are described in detail hereinbelow in context with specific examples of host cells.
  • the transformed host cell can be a eukaryotic cell, such as, for example, a mammalian cell, an insect cell, a plant cell, a yeast cell and a protozoa cell, or alternatively, the cell can be a bacterial cell.
  • a eukaryotic cell such as, for example, a mammalian cell, an insect cell, a plant cell, a yeast cell and a protozoa cell, or alternatively, the cell can be a bacterial cell.
  • the nucleic acid construct(s) according to the present invention can be a shuttle vector, which can propagate both in E. coli (wherein the construct comprises an appropriate selectable marker and origin of replication) and be compatible for expression in eukaryotic host cells.
  • the nucleic acid construct(s) according to the present invention can be, for example, a plasmid, a bacmid, a phagemid, a cosmid, a phage, a virus or an artificial chromosome.
  • Suitable mammalian expression systems include, but are not limited to, pcDNA3, pcDNA3.1( ⁇ ), pZeoSV2( ⁇ ), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, which are available from InvitrogenTM Corporation (Carlsbad, Calif. USA), pCI which is available from PromegaTM Corporation (Madison Wis. USA), pBK-RSV and PBK-CMV which are available from Stratagene® (La Jolla, Calif. USA), pTRES which is available from Clontech® Laboratories, Inc. (Mountain View, Calif. USA), and their derivatives.
  • Insect cell cultures can also be utilized to express the nucleic acid sequences of the present invention.
  • suitable insect expression systems include, but are not limited to the baculovirus expression system and its derivatives which are commercially available from numerous suppliers such as maxBacTM (InvitrogenTM Corporation, Carlsbad, Calif. USA) BacPakTM (Clontech® Laboratories, Inc. Mountain View, Calif. USA), or Bac-to-BacTM (InvitrogenTM/Gibco®, Carlsbad, Calif. USA).
  • plant cell can refer to plant protoplasts, cells of a plant tissue culture, cells of plant derived tissues or cells of whole plants.
  • nucleic acid constructs into plant cells. Such methods rely on either stable integration of the nucleic acid construct or a portion thereof into the genome of the plant cell, or on transient expression of the nucleic acid construct in which case these sequences are not stably integrated into the genome of the plant cell.
  • the Agrobacterium system includes the use of plasmid vectors that contain defined DNA segments that integrate into the plant genomic DNA. Methods of inoculation of the plant tissue vary depending upon the plant species and the Agrobacterium delivery system. A widely used approach is the leaf disc procedure, see for example, Horsch et al. in Plant Molecular Biology Manual A5, Kluwer Academic Publishers, Dordrecht (1988) p. 1-9. A supplementary approach employs the Agrobacterium delivery system in combination with vacuum infiltration. The Agrobacterium system is especially viable in the creation of stably transformed dicotyledenous plants.
  • suitable plant promoters which can be utilized for plant cell expression of the first and second nucleic acid sequences, include, but are not limited to CaMV 35S promoter, ubiquitin promoter, and other strong promoters which can express the nucleic acid sequences in a constitutive or tissue specific manner.
  • Plant viruses can also be used as transformation vectors. Viruses that have been shown to be useful for the transformation of plant cell hosts include CaV, TMV and BV. Transformation of plants using plant viruses is described in U.S. Pat. No. 4,855,237 (BGV), EPA 67,553 (TMV), Japanese Published Application No. 63-14693 (TMV), EPA 194,809 (BV), EPA 278,667 (BV); and Gluzman, Y. et al., Communications in Molecular Biology: Viral Vectors, Cold Spring Harbor Laboratory, New York, pp. 172-189 (1988). Pseudovirus particles for use in expressing foreign DNA in many hosts, including plants, is described in WO 87/06261.
  • the constructions can be made to the virus itself.
  • the virus can first be cloned into a bacterial plasmid for ease of constructing the desired viral vector with the nucleic acid sequences described above. The virus can then be excised from the plasmid. If the virus is a DNA virus, a bacterial origin of replication can be attached to the viral DNA, which is then replicated by the bacteria. Transcription and translation of this DNA will produce the coat protein which will encapsidate the viral DNA.
  • the virus is an RNA virus, the virus is generally cloned as a cDNA and inserted into a plasmid. The plasmid is then used to make all of the constructions. The RNA virus is then produced by transcribing the viral sequence of the plasmid and translation of the viral genes to produce the coat protein(s) which encapsidate the viral RNA.
  • Yeast cells can also be utilized as host cells by the present invention.
  • Numerous examples of yeast expression vectors suitable for expression of the nucleic acid sequences of the present invention in yeast are known in the art and are commercially available. Such vectors are usually introduced in a yeast host cell via chemical or electroporation transformation methods well known in the art.
  • Commercially available systems include, for example, the pYESTM (InvitrogenTM Corporation, Carlsbad Calif., USA) or the YEXTM (Clontech® Laboratories, Mountain View, Calif. USA) expression systems.
  • the nucleic acid construct when expressed in eukaryotic expression systems such as those described above, preferably includes a signal peptide encoding sequence such that the polypeptides produced from the first and second nucleic acid sequences are directed via the attached signal peptide into secretion pathways.
  • the expressed polypeptides in mammalian, insect and yeast host cells, can be secreted to the growth medium, while in plant expression systems the polypeptides can be secreted into the apoplast, or directed into a subcellular organelle.
  • a bacterial host can be transformed with the nucleic acid sequence via transformation methods well known in the art, including for example, chemical transformation (e.g., CaCl 2 ) or electroporation.
  • transformation methods well known in the art, including for example, chemical transformation (e.g., CaCl 2 ) or electroporation.
  • bacterial expression systems which can be utilized to express the nucleic acid sequences of the present invention are known in the art.
  • Commercially available bacterial expression systems include, but are not limited to, the pETTM expression system (Novagen®, EMB Biosciences, San Diego, Calif. USA), pSETM expression system (InvitrogenTM Corporation, Carlsbad Calif., USA) or the pGEXTM expression system (Amersham Biosciences, Piscataway, N.J. USA).
  • bacterial expression is particularly advantageous since the expressed polypeptides form substantially pure inclusion bodies readily amenable to recovery and purification of the expressed polypeptide.
  • this invention provides a preparation of bacterial-expressed inclusion bodies which are composed of over 30%, preferably over 50%, more preferably over 75%, most preferably over 90% by weight of the fusion protein or a mixture of fusion proteins of the present invention.
  • inclusion bodies which are composed of over 30%, preferably over 50%, more preferably over 75%, most preferably over 90% by weight of the fusion protein or a mixture of fusion proteins of the present invention.
  • the isolation of such inclusion bodies and the purification of the fusion protein(s) therefrom are described in detail in the Experimental Details section which follows. Bacterial expression of the fusion protein(s) can provide high quantities of pure and active forms of fusion proteins.
  • the expressed fusion proteins form substantially pure inclusion bodies which are readily isolated via fractionation techniques well known in the art and purified via for example denaturing-renaturing steps.
  • the fusion proteins of the invention may be renatured and refolded in the presence of a MHC-restricted peptide, which is either linked to, co-expressed with or mixed with other polypeptides of the invention and being capable of binding the single chain MHC class I polypeptide. As is further described in the examples section, this enables to generate a substantially pure MHC class I-antigenic peptide complex which can further be purified via size exclusion chromatography.
  • the CMV peptide used for refolding can be co-expressed along with (as an independent peptide) or be fused to the scHLA-A2 chain of the MHC Class I molecule in the bacteria.
  • the expressed fusion protein and peptide co-form inclusion bodies which can be isolated and utilized for MHC class I-antigenic peptide complex formation.
  • the scHLA-A2/SS1 was constructed as previously described by linking the C-terminus of scHLA-A2 to the N-terminus of the SS1 scFv via a short linker ASGG (SEQ ID NO:4) (15).
  • the MHC-restricted peptide was fused with the peptide linker GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:6) to the N-terminus of the scHLA-A2/SS1 (scFv) molecule by a PCR overlap extension reaction with the primers: 5′M1-5′GGAAGCGTTGGCGCATATGGGCATTCTGGGCTTCGTGTTTACC CTGGGCGGAGGAGGATCCGGTGGCGGAGGTTCAGGAGGCGGTGGATCGA TCCAGCGTACTCCAAAG3′(SEQ ID NO: 13) and 3′VLscSS1-5′GCAGTAAGGAATTCTCATTATTTTATTTCCAACTTTGT3′(SEQ ID NO: 14)
  • a silence mutation was inserted at the linker sequence, this change in sequence creates a BamH1 restriction site
  • PCR products were sub-cloned to TA cloning vector (pGEM-T Easy Vector, PromegaTM Corporation, Madison, Wis. USA) and subsequently to a T7 promoter-based expression vector (PRB) using the NdeI and EcoRI restriction sites.
  • TA cloning vector pGEM-T Easy Vector, PromegaTM Corporation, Madison, Wis. USA
  • PRB T7 promoter-based expression vector
  • M1/scHLA-A2/SS1 (scFv) was used as a template for ligation with dsDNA primer.
  • the M1/scHLA-A2/SS1 (scFv) (in PRB plasmid) was digested with NdeI and BamHI, and the plasmid fraction was ligated to dsDNA primer containing the CMV peptide sequence and the extension of the linker sequence 5′CMVcovLL (cassette):5′TATGAACCTGGTGCCGATGGTCGCGACCGT TGGAGGTGGCGGTTCTGGCGGAGGAG-3′ (SEQ ID NO: 15) and 3′CMVcovLL (cassette):5′GATC CTCCTCCGCCAGAACCGCCACCTCCAACGGTCGCGACCATCGGCACCAGG TTCA3′(SEQ ID NO:16).
  • the annealing of the primers (5′CMVcovLL (cassette) and 3′CMVcovLL (cassette)) was performed by incubating the primers at 95° C. for 2 min followed by 1 h incubation at room temperature.
  • the ligation product was transformed to E-coli DH5 ⁇ for plasmid amplification. Plasmid was purified by QIAGEN® MiniprepTM, DNA isolation kit (Qiagen®, Inc., Valencia, Calif. USA) and samples were set for sequence analysis.
  • Compound A was expressed in E - coli LB21 ( ⁇ DE3) cells (Novagen®, Madison, Wis. USA) as inclusion bodies.
  • Inclusion bodies were purified from cell pellet by cell disruption with 0.2 mg/ml of lysozyme followed by the addition of 2.% Triton® X-100 (Octylphenolpoly[ethyleneglycolether] x , Roche Diagnostics GmbH, Roche Applied Science, Mannheim, Germany) and 0.5M NaCl.
  • the pellets of the inclusion bodies were collected by centrifugation (13,000 rpm, 60 min at 4° C.) and washed three times with 50 mM Tris buffer pH 7.4 containing 20 mM EDTA.
  • the isolated and purified inclusion bodies were solubilized in 6M Guanidine HCl pH 7.4, followed by reduction with 65-mM DTE.
  • Solubilized and reduced inclusion bodies were refolded by a 1:100 dilution into a redox-shuffling buffer system containing 0.1-M Tris, 0.001M EDTA, 0.5-M Arginine, and 0.09-mM Oxidized Glutathione, pH 9, and incubation at 10° C. for 24 h.
  • the protein was dialyzed against 150-mM Urea, 20-mM Tris, pH 8, followed by purification of the soluble Compound A by ionexchange chromatography on a Q-Sepharose® column (7.5 mm I.D 60 cm) (Sigma-Aldrich, Inc., St. Louis, Mo. USA), applying a salt (NaCl) gradient. Peak fractions containing Compound A were then subjected to buffer exchange with PBS.
  • the scHLA-A2/SS1 was constructed as previously described by linking the C-terminus of scHLA-A2 to the N-terminus of the SS1 scFv via a short linker ASGG (SEQ ID NO:15).
  • the MHC-restricted peptide and the peptide linker GGGGSGGGGSGGGGS were fused to the N-terminus of the scHLA-A2/SS1 (scFv) molecule by a PCR overlap extension reaction with the primers 5′-Nde-209B2M:5′GGAAGCGTTGGCGCATATGATCATGGACCAGGTT CCGTTCTCTGTTGGCGAGGAGGGTCCGGTGGCGGAGGTTCAGGAGGCGGTG GATCGATCCAGCGTACTCCAAAG3′(SEQ ID NO: 17) And the 3′VLscSS1-5′GCAGTAAGG AATTCTCAT TATTTTATTTCCAACTTTGT3′(SEQ ID NO:18).
  • 209cov/scHLA-A2/SS1 (scFv) molecule was used as a template for the construction of Compound B.
  • the CMV peptide NLVPMVATV (SEQ ID NO: 4) was introduced into the 209cov/scHLA-A2/SS1 (scFv) sequence (exchanging the 209 peptide) by PCR reaction using the primers 5′GGAAGCGTTGGCGCATATGG GCATTCTGGGCTTCGTGTTTACCCTGGGCGAGGAGGATCCGGTGGCGGAGGTTCAGGAGGCGGTGGA TCGATCCAGCGTACTCCAAAG3′(SEQ ID NO: 17) and the 3′VLscSS15′GCAGTAAGGAATTCTCATTATTTTAT TTCCAACTTTGT3′(SEQ ID NO: 18).
  • the MI 58-66 peptide was fused to the N-terminus of scHLA-A2/SS1 (scFv) fusion protein through a short 15 amino acid linker by overlapping PCR reaction with the 5′M1-linker primer: 5′GGAAGCGTTGGCGCATATGGGCATTCTGGGCTTCGTGTTTACCCTGGGCGG AGGAGGATCCGGTGGCGGAGGTTCAGGAGGCGGTGGATCGATCCAGCGTACTCCAAAG3′(SEQ ID NO: 13) and the 3′VLscSS1-5′GCAGTAAGGAATTCTCAT TATTTTATTTCCAACTTTGT3′(SEQ ID NO: 14).
  • PRB plasmid was used as a template containing the scHLA-A2/SS1 (scFv) sequence.
  • the expression and purification protocols of the M1-cov/scHLA-A2/SS1 (scFv) fusion protein were identical to the expression and purification protocols of Compound A. All the methods used to analyse the biochemical and biological properties of the M1-cov/scHLA-A2/SS1 (scFv) fusion protein were identical to the methods used to analyse the activity of Compound A.
  • Cells were incubated with Compound A (60 min at 4° C. in 100 ⁇ l, 10 ⁇ g/ml), washed and incubated with the anti-HLA-A2 MAb BB7.2 (60 min at 4° C., 10 ⁇ l/ml). The cells were washed and incubated with anti-mouse FITC (60 min at 4° C., 10 ⁇ l/ml) that served as a secondary antibody. The cells were subsequently washed and analyzed by a FACS caliber flow cytometer (Becton-Dickinson, San Jose, Calif. USA).
  • Immunoplates (Falcon®, Becton-Dickinson Labware, Franklin Lakes, N.J. USA) were coated with 10 ⁇ g/ml of purified bacterially produced recombinant mesothelin (O/N at 4° C.). The plates were blocked with PBS containing 2% skim milk and then incubated with various concentrations of Compound A (60 min at RT) and washed three times with PBS.
  • Binding was detected using the anti-HLA-conformational-dependent antibody W6/32 (60 min, RT, 1 ⁇ g/ml), plates were washed three times with PBS and incubated with anti-mouse IgG-peroxidase (60 min, RT, 1 ⁇ g/ml). The reaction was developed using TMB (DAKO) and terminated by the addition of 50 ⁇ l H 2 SO 4 2N. Anti-mesothelin antibody (K1) was used as a positive control. The immunoplates were analyzed by ELISA reader using 450 nm filter (Anthos 2001TM, Anthos Labtech, Salzburg, Austria).
  • Cytotoxicity was determined by S 35 -methionine release assays.
  • Target cells were cultured in culture plates in RPMI 10% FCS Methionine free for 2 h, followed by incubation overnight with 15 ⁇ Ci/ml of S 35 methionine (NEN). The target cells were harvested by trypsinization and washed twice with 40 ml RPMI 10% FCS. The target cells were plated in 96-well plates (5 ⁇ 10 3 cells per well) in RMPI+10% FCS and incubated overnight at 37° C., 5% CO 2 .
  • Target cells were incubated with different concentrations of Compound A fusion proteins for 2 h, effector CTL cells were added at different target: effector ratios and the plates were incubated for 8-12 h at 37° C., 5% Co 2 . Following incubation, S 35 -methionine release from target cells was measured in a 25 ⁇ l sample of the culture supernatant. All assays were performed in triplicate, lysis was calculated directly: ([experimental release ⁇ spontaneous release]/[maximum release ⁇ spontaneous release]) ⁇ 100. Spontaneous release was measured as S 35 methionine released from target cells in the absence of effector cells, and maximum release was measured as S 35 -methionine released from target cells lyzed by 0.05M NaOH.
  • A431 and A431K5 cells were maintained in RPMI medium containing 10% FCS, L-glutamine and penicillin/streptomycin.
  • the A431K5 cell line is a human epidermoid carcinoma A431 cell line stably transfected with Mesothelin, the transfected cells were maintained with 700 ⁇ g/ml G418 (Gibco-BRL®, InvitrogenTM Inc., Carlsbad, Calif. USA).
  • CTL's with specificity for CMV pp 65 epitope were kindly provided by Dr Ditmar Zehn (Charitee, Berlin).
  • the CTL's were expanded by incubation with peptide pulsed, radiated (4000rad) PBMC's from a healthy HLA-A2 positive donor and were maintained in AIMV medium+8.9% FCS+50 ⁇ M-2-mercaptoethanol+penicillin/streptomycin 1 ⁇ 10 5 U/L.
  • This construct was analyzed in detail for its biochemical and biological activity and was found to be functional in-vitro and in-vivo (15).
  • a 9 amino acids peptide derived from the CMV pp65 protein (NLVPMVATV) (SEQ ID NO:4) was fused to the N-terminus of the scHLA-A2/SS1 (scFv) fusion protein via 20 amino acids linker GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:6) ( FIG. 1B ).
  • Compound A was constructed in two steps: First a covalent fusion protein termed M1/scHLA-A2/SS1 (scFv) was constructed by overlap extension PCR.
  • influenza M158-66 peptide GILGFVFTL SEQ ID NO:21
  • a 15 amino acid linker was fused to the N-terminus of the scHLA-A2/SS1 (scFv) fusion protein.
  • a new, unique restriction site BamHI
  • PRB plasmid containing the M1/scHLA-A2/SS1 (scFv) full sequence was digested with NdeI and BamHI restriction enzymes. This digestion produced two fragments.
  • One fragment contains the peptide and part of the linker sequence
  • the second fragment contains the plasmid, part of the linker and the scHLA-A2/SS1 (scFv) sequence.
  • the fragment which contains the plasmid, part of the linker and the scHLA-A2/SS1 (scFv) sequence was then ligated to dsDNA primer that codes the CMV pp65 peptide sequence and an extension of the linker sequence ( FIG. 1B ).
  • the new plasmid was transformed to E - coli DH5 ⁇ cells and positive colonies were sent to DNA sequencing ( FIG. 2 ).
  • Compound A was expressed in E. coli BL21 cells and, upon induction with isopropyl ⁇ -D-thiogalactoside, large amounts of recombinant protein accumulated in intracellular inclusion bodies.
  • SDS/PAGE analysis of isolated and purified inclusion bodies revealed that Compound A with the correct size constituted 80-90% of the total inclusion bodies mass ( FIG. 3A ).
  • the isolated solubilized inclusion bodies were reduced and refolded in-vitro in a redox-shuffling buffer.
  • Monomeric soluble fusion proteins (Compound A) were purified by ion-exchange chromatography on Q-sepharose®.
  • SDS/PAGE analysis of Compound A revealed a highly purified monomeric molecule with the expected size of 72 KDa ( FIG. 3B ).
  • the recombinant mesothelin was immobilized to immunoplates.
  • the binding of Compound A was monitored by using conformation sensitive mAb W6/32, this antibody recognizes MHC molecules that are folded correctly with a peptide in its groove.
  • the binding of Compound A to recombinant mesothelin was dose-dependent. This suggests that the two functional domains of Compound A, the scFv (SS1) domain and the peptide/scHLA-A2 domain are folded correctly.
  • the scFv (SS1) domain of the fusion protein is in active form and can specifically bind mesothelin.
  • HLA-A2 negative A431K5 cells were used, which are human epidermoid carcinoma A431 cells that were stably transfected with mesothelin.
  • the parental A431 human epidermoid carcinoma cells which are mesothelin-negative and HLA-A2-negative are used as negative control.
  • the binding of Compound A to the target cells was monitored with anti-HLA-A2 mAb BB7.2 as primary antibody followed by a FITC labeled secondary antibody.
  • a mesothelin anti-mAb K1 was used to test the expression levels of mesothelin.
  • A431K5 cells express high levels of mesothelin, whereas the parental A431 cells do not express the target antigen.
  • the cell lines A431 and A431K5 were also tested for the expression of HLA-A2 using HLA-A2 specific antibody (BB7.2), both cell lines were HLA-A2 negative. However, when A431K5 cells were pre-incubated with Compound A, they were positively stained with the HLA-A2 specific antibody BB7.2 ( FIG. 5B ). Antigen-negative A413 cells were not affected.
  • the specific binding of Compound A to A431K5 but not to A431 cells further indicates that the binding is exclusively depended on the interaction of the targeting scFv domain of the fusion with mesothelin and that the fusion protein can bind its target antigen as natively expressed on the surface of cells.
  • S 35 -Methionine release assay was performed using HLA-A2-negative mesothelin-transfected A431K5 cells, and the HLA-A2-negative mesothelin-negative A431 parental cells.
  • cytotoxicity assay was performed using HLA-A2-positive JY cells that were radiolabeled with MetS 35 and laded with the CMV peptide NLVPMVATV (SEQ ID NO:4).
  • A431K5 cells which are human epidermoid carcinoma A431 cells that were stably transfected with mesothelin and are HLA-A2 negative.
  • the parental A431 human epidermoid carcinoma cells were used which are mesothelin-negative and HLA-A2 negative.
  • the binding of Compound B to the target cells was monitored by anti-HLA-A2 mAb BB7.2 as primary antibody followed by a FITC labeled secondary antibody.
  • anti-HLA-A2 mAb BB7.2 as primary antibody followed by a FITC labeled secondary antibody.
  • a commercial anti-mesothelin mAb K1 was used.
  • FIG. 10A-B A431K5 cells express high levels of mesothelin, whereas the parental A431 cells do not express mesothelin.
  • the cell lines A431 and A431K5 were also tested for their expression of HLA-A2 using HLA-A2 specific anti body (BB7.2), both cell lines were HLA-A2 negative.
  • A431K5 cells were pre-incubated with Compound B, they were positively stained with the HLA-A2 specific antibody BB7.2 whereas control A431 cells were not stained ( FIG. 10C-D ).
  • the specific binding of Compound B to A431K5 cells but not to A431 cells indicate that binding is exclusively depended on the interaction of the targeting scFv domain with mesothelin.
  • the average specific killing of the JY cells by the CMV-specific CTLs was around 45-50% using an E:T ratio of 10:1 (data not shown).
  • Compound B effectively mediated the killing of the A431K5 cells (mesothelin-positive HLA-A2-negative), this specific killing was 66% in comparison with peptide-loaded JY cells ( ⁇ 150% compared to JY cells).
  • the target A431K5 cells were incubated with the CMV-specific CTLs alone without preincubation with Compound B or when the target cells were mesothelin-negative (A431 cells), with or without preincubation with Compound B, no cytotoxic activity was observed.
  • the M1-cov/scHLA-A2/SS1 (scFv) fusion protein was constructed by overlap extension PCR reaction in which the Influenza M1 58-66 peptide and a 15 amino acid linker GGGGSGGGGSGGGGS were fused to the N-terminus of the scHLA-A2/SS1 (scFv) fusion protein ( FIG. 13 ).
  • the PCR product was ligated to TA-cloning vector (p-GEM, Promega), transformed to E - coli DH5 ⁇ cells. Positive colonies were selected and the insert was isolated using EcoRI and NdeI.
  • the insert was ligated to PRB expression vector and transformed to E - coli DH5 ⁇ cells. Positive colonies were sent to DNA sequencing ( FIG. 14 ).
  • the M1-cov/scHLA-A2/SS1 (scFv) fusion protein was expressed in E. coli BL21 cells and, upon induction with isopropyl ⁇ -D-thiogalactoside, large amounts of recombinant protein accumulated in intracellular inclusion bodies.
  • SDS/PAGE analysis of isolated and purified inclusion bodies revealed that the M1-cov/scHLA-A2/SS1 (scFv) fusion protein with the correct size constituted 80-90% of the total inclusion bodies mass ( FIG. 15A ).
  • the isolated solubilized inclusion bodies were reduced and refolded in vitro in a redox-shuffling buffer.
  • Monomeric soluble fusion proteins (M1-cov/scHLA-A2/SS1 (scFv)) were purified by ion-exchange chromatography on Q-sheparose. SDS/PAGE analysis of the M1-cov/scHLA-A2/SS1 (scFv) fusion proteins revealed a highly purified monomeric molecule with the expected size of 72 KDa ( FIG. 15B ).
  • scFv M1-cov/scHLA-A2/SS1
  • the binding of the M1-cov/scHLA-A2/SS1 (scFv) fusion protein to the target cells was monitored by anti-HLA-A2 mAb BB7.2 as primary antibody followed by a FITC-labeled secondary antibody.
  • anti-HLA-A2 mAb BB7.2 anti-HLA-A2 mAb
  • a commercial anti mesothelin mAb K1 was used.
  • A431K5 cells express high levels of mesothelin, whereas the parental A431 cells do not express mesothelin.
  • the cell lines A431 and A431K5 were also tested for their expression of HLA-A2 using HLA-A2 specific antibody (BB7.2), both cell lines were HLA-A2-negative.
  • M1-cov/scHLA-A2/SS1 (scFv) fusion protein To test the ability of the M1-cov/scHLA-A2/SS1 (scFv) fusion protein to mediate the killing of HLA-A2-negative mesothelin-positive cells by HLA-A2-restrictive M158-66 specific CTLs, S 35 -Methionine release assay using HLA-A2-negative mesothelin-transfected A431K5 cells was performed. As shown in FIG. 18 , M1-cov/scHLA-A2/SS1 (scFv) fusion protein did not mediate the lysis of A431K5 cells (mesothelin-positive HLA-A2-negative).
  • the scHLA-A2/SS1 (scFv) bearing the M158-66 peptide in its groove mediated the killing of mesothelin-positive target cells by the HLA-A2-restricted M158-66 specific CTLs.
  • This study demonstrates the ability to target covalently linked peptide/scMHC/scFv fusion protein to tumor cells can render HLA-A2-negative cells susceptible to lysis by the relevant HLA-A2-restricted CTLs.
  • this strategy has two major advantages. First, it takes advantage of the use of recombinant Ab fragments that can localize on those malignant cells that express a tumor marker, usually associated with the transformed phenotype (such as growth factor receptors and/or differentiation antigens), with a relatively high degree of specificity.
  • this strategy has the ability to recruit a particular population of highly reactive cytotoxic T-cells specific to a preselected, highly antigenic peptide epitope present in the targeted MHC-peptide complex, such as viral-specific T-cell epitopes.
  • This platform approach generates multiple molecules with many tumor-specific scFv fragments that target various tumor specific antigens, combined with the ability to target many types of MHC-peptide complexes carrying single, preselected, and highly antigenic peptides derived from tumor, viral, or bacterial T-cell epitopes.
  • the present invention relates to construction of a fusion protein in which the CMV pp65 derived (NLVPMVATV) fused to the N-terminus of the scHLA-A2/SS1 (scFv) molecule and its biochemical and biological characteristics.
  • the two domains of the new fusion protein can refold in vitro to form correctly folded molecules with the peptide within the HLA-A2 groove and an active targeting domain (scFv) that can specifically bind its target antigen.
  • this fusion protein had successfully mediated the lysis of HLA-A2-negative mesothelin-positive tumor cells by HLA-A2-restricted CTLs.
  • Tumor progression is often associated with the secretion of immune-suppressive factors and/or the down-regulation of MHC class I antigen-presentation functions (2). Even when a specific CTL response is demonstrated in patients, this response is low because the anti-tumor CTL population is rare, very infrequent, and in some cases the CLTs are not functional or anergic (26). Moreover, it is well-established that the number of MHC-peptide complexes on the surface of tumor cells that present a particular tumor-associated peptide is low (27). As shown herein, the new strategy overcame these problems. First, the tumor cells are coated with MHC-peptide complexes independent of their endogenous MHC expression.
  • tumor specific antigens that are usually part of the tumor phenotype (such as growth factor receptors and differentiation antigens) prevent the down regulation of those antigens and prolong the efficiency of the treatment.
  • tumor specific antigens that are usually part of the tumor phenotype (such as growth factor receptors and differentiation antigens) prevent the down regulation of those antigens and prolong the efficiency of the treatment.
  • the effector domain of the fusion protein the MHC-peptide complex can recruit specific populations of CTLs depending on the peptide harboring the MHC groove.

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